A fully defined system for DNA packaging has been developed for Bacillus subtilis bacteriophage 029 that approximates in vivo assembly in efficiency. ATP, the DNa packaging protein gp16, purified proheads and DNA-gp3 comprise the complete system. A small RNA of 174 nucleotides is essential for DNA-gp3 packaging and is associated with the prohead vertex. The RNA can be detached and reattached, with concomitant loss and restoration of competence to package DNA-gp3 in the defined system. The gp16, overproduced in Escherichia coli, contains ATP-binding sequences, and the binding and hydrolysis of ATP by gp16 is both prohead and DNA-gp3 dependent. Approximately one molecule of ATP is used to package two base-pairs of 029 DNA. We have proposed a new model for prohead assembly in which RNa initiates assembly by binding the connector protein gp10 to form pentameric protomers. These protomers assemble into a particle by the addition of the scaffold and shell proteins. Further recruitment of shell protein occurs with the loss of gp10. The insertion of a connector dodecamer then forms the portal vertex and permits the final assembly of the neck- tail complex. The three-dimensional structure of the 029 prohead, packaging intermediates and the DNA-filled head will be studied by single crystal X-ray diffraction and by image analysis of electron micrographs of frozen-hydrated samples. These studies will complement our study of the morphopoietic properties of prohead RNA and the scaffold and connector proteins in head assembly. Purified RNa and prohead proteins and a number of unusual particles produced in nonpermissive infections by temperature-sensitive (ts) mutants will be used to confirm our assembly model and ultimately to construct the competent prohead in vitro. We will study the role of the shell protein (gp8) during DNA-gp3 packaging. We will study conformational changes of gp8, functional contact between the shell protein and DNA-gp3, and a direct role of gp8 that may involve ATP hydrolysis during DNA-gp3 packaging. We will study quantized packaging of DNA-gp3 by altering the packaging environment, by the use of proheads formed by ts8 mutants, and by the study of particles that package genomic fragments. We will investigate the hypothesis that RNA may add stability and orientation to the DNA- gp3 complex during packaging. We are studying prohead assembly, DNA encapsidation, and the mechanisms of form determination in the most efficient in vitro DNA packaging and viral assembly system known.